1. Field of the Invention
The species Acidovorax avenae, formerly classified as Pseudomonas avenae, contains different subspecies pathogenic to several hosts including oat, corn, millet, wheat, sugarcane, rice, orchid, konjaci, and Cucurbitaceae plants including melon, watermelon, and squash. This invention relates to novel PCR primers which can be used to detect plant pathogenic Acidovorax avenae species and to distinguish among the pathogenic subspecies and strains, particularly to distinguish (1) A. avenae from other bacteria, (2) A. avenae subsp. citrulli (infects only Cucurbitaceae) from other subspecies and (3) rice strains of A. avenae subsp. avenae from other strains originating from other plants as well as from other subspecies of A. avenae. 
2. Description of the Relevant Art
The bacterium A. avenae subsp. avenaecauses bacterial stripe of rice, leaf blight of oats, red stripe disease of sugarcane and millet, and brown stripe of Setaria italica. The organism was originally described as Pseudomonas avenae by Manns in 1909 (Ohio Agric. Exp. Stn. Res. Bull. 210: 91-167) as the causal agent of leaf blight on oats in Ohio. Another organism, P. alboprecipitans, described by Rosen in 1922 on foxtail (Ann. Mo. Bot. Gard. 9:333-402) and on corn in 1926 (Phytopathology 16:241-267) was later shown to be synonymous with P. avenae (Schaad et al. 1975. Int J. Syst Bacteriol. 25:133-137). On rice, the organism has been previously referred to as P. setariae or P. panici (Goto. 1964. Bull. of Faculty of Agriculture, Shizuoka University 14: 3-10) and on sugarcane as P. rubrilineans (Martin et al. 1989.), the causal agent of red stripe diseases of sugarcane (C. Ricaud et al. (Eds), Elsevier, The Netherlands, pp. 80-95). Acidovorax avenae subsp. citrulli causes bacterial blight and fruit blotch of watermelon and melon. The organism was originally described by Schaad et al. (1978. Int. J. Syst. Bacteriol. 28:117-125) in plant introduction breeding lines of watermelon in Georgia as P. pseudoalcaligenes subsp. citrulli. A closely related organism, A. avenae subsp. cattleyae occurs on orchids (Ark and Thomas. 1946. Phytopathology 36:695-698) and A. konjaci (Goto, M. 1983. Int J. Syst. Bacteriol. 33:539-545) occurs on konjac (Amorphophalus konjaci Koch).
As the host range of A. avenae subsp. avenae is wide to monocotyledonous and dicotyledonous plants, it has been thought to be polyxenic. However, individual strains of the pathogen infect only one or a few host species (Kadota, I. 1996. Bulletin of the Hokuriku National Agricultural Experiment Station 38:113-171; Nishiyama et al. 1979. Ann. Phytopathol. Soc. Jpn. 45:25-31). For example, rice strains can infect rice but not other monocotyledonous plants including finger millet, whereas finger millet strain cannot infect rice plants. Furthermore, the heterogeneity of rice strains and other plant strains of the bacterium has been suggested based on the biochemical characteristics and the protein profiles of the cells. Even though the reclassification of previous P. avenae strains to A. avenae subsp. avenae has been widely accepted, a certain degree of pathogenic, serological, and genomic, as well as phenotypic, heterogeneity still exists among reported strains of A. avenae subsp. avenae subspecies. Pathogenicity of A. avenae subsp. avenae strains specific for rice particularly differs from non-rice strains of A. avenae subsp. avenae. It has been thought, therefore, that rice strains of A. avenae subsp. avenae are different from other plant strains. However, hitherto there is no sensitive and specific means which can detect all the strains of A. avenae subsp. avenae which have originated from different hosts and thus satisfy the current taxonomic situation at the subspecies level.
Phylogenetically, A. avenae is included in the new xe2x80x9cacidovoransxe2x80x9d DNA-rRNA homology group (Willems et al. 1992. Int J. Syst Bacteriol. 42:107-119). This group contains many pathogens previously classified in the non-fluorescent pseudomonad group, including P. avenae Manns 1909 (Ohio Agric. Exp. Stn. Res. Bull. 210:91-167), P. cattleyae Savulescu 1947 (Pavarino. 1911. Atti Accad. Lincei 20:233-237), P. pseudoalcaligenes subsp. citrulli (Schaad et al. 1978, supra), P. pseudoalcaligenes subsp. konjaci (Goto, 1983, supra) and P. rubrilineans (Lee et al. 1925. Red Stripe Disease. Pamphlet of Hawaiian Sugar Planters Association). Recently, all of these bacteria have been reclassified as subspecies of A. avenae, i.e., A. avenae subsp. avenae, A. avenae subsp. cattleyae, and A. avenae subsp. citrulli, on the basis of the results of DNA-DNA hybridization, DNA-rRNA hybridizations, polyacrylamide gel electrophoresis of whole-cell proteins, and a numerical analysis of carbon assimilation tests (Willems et al., supra).
A. avenae subsp. avenae is one of the major seedborne bacterial pathogens of rice. Seeds infested with the pathogen are important sources of primary inoculum and a means of dissemination to new areas. Therefore, sensitive and simple detection methods are needed to screen seed lots. Presently, the only reliable method to identify the A. avenae pathogens is to isolate them on agar media and confirm their presumptive identification by time consuming and expensive pathogenicity tests. Although a few methods have been used for the detection of A. avenae subsp. avenae, none has been optimized for selectively detecting the bacterium from rice seeds. Serological methods and a rapid pathogenicity test have been used for routine detection of this pathogen in rice seeds (Kadota et al., supra; Shakya. 1987. Korean J. Plant Path. 3:300; Zeigler et al. 1989. Intl. Rice Res. Newsletter 14:27-28). However, in addition to the issues of specificity and sensitivity, serological techniques have the distinct disadvantage of not being able to provide information on viability and pathogenicity of the seedborne inoculum. In rice, A. avenae subsp. avenae can be recovered from both diseased, or asymptomatic, apparently healthy seeds. However, identification is difficult, because the organism is frequently overgrown by other bacterial pathogens of rice, such as Pantoea (Erwinia) herbicola, Burkholdera (Pseudomonas) glumae, B. fuscovaginae, or P. syringae pv. syringae. These pathogens are recognized as producing distinct disease symptoms, but field diagnosis is very difficult. In such cases, detection and identification is based on pathogen isolation, through the use of semiselective media, and on pathogenicity tests which discriminate between the various pathogens. However, the presence of coexisting epiphytes such as P. fulva, P. corrugata, P. putida, and P. fluorescens in/on rice seed makes isolation difficult. Furthermore, rice seedlings are difficult to grow and symptoms on seedlings are often non-discriminating. Several methods of identification of A. avenae subsp. avenae have been proposed including growth and isolation on semiselective agar media and serology (Shakya, supra). None have achieved much success. The disease cycle, therefore, has not been clarified, because the symptoms are masked after the four or five leaf stage and no adequate techniques are available for monitoring the pathogen in rice plants. Further, there is currently no reliable, routine method available for detecting A. avenae in rice seeds. More sensitive and specific methods are needed to confirm the identification, especially in seed health evaluations.
Similar problems exist for watermelon and melons. Although originally described as a seedling disease, watermelon fruit blotch has emerged as a serious disease of mature fruit. Complete losses of production fields often occur due to fruit rot. Like most seedborne bacteria, control is based primarily on seed health testing. Because of the seriousness of watermelon fruit blotch, nearly all watermelon seed lots must be assayed for A. avenae subspec. citrulli. This involves soaking 30,000 seeds and attempting to isolate the organism on agar media or inoculating plants with the seed soakate. Neither method is very efficient and both are relatively expensive. Little or no resistance to the pathogen exists.
Bacterial stripe causes great losses in rice seedling beds throughout Asia. Thus far, the disease has not been observed in the U.S. However, the potential risk of the dissemination of the bacterial stripe pathogen in international exchange of germplasm of rice and corn is a serious concern. There is presently no way to separate infected seeds and contaminated seeds. Therefore, there is a need to develop reagents and methods for detecting A. avenae subspecies specifically, rapidly, and directly from biological samples. Such methods and reagents are valuable tools for monitoring natural disease spread, tracking the seedborne bacteria in field studies, and detecting the presence of the bacterium in seed lots entering A. avenae-free areas.
Polymerase chain reaction (PCR) has been shown to be highly sensitive and the method is commonly used to detect and identify bacteria. A PCR method has been described for detecting the pathogen A. avenae subsp. citrulli, in seeds; however, the primers are not unique and the method has not gained industry acceptance (Minsavage et al. 1995. Ann. Mtg. Amer. Phytopath. Abstract 379). Thus, there exists a need for specific primers and methods capable of specifically identifying and differentiating pathogenic A. avenae subspecies and strains.
We have discovered oligonucleotide sequences which are capable of amplifying DNA fragments specific for identifying the two closely related pathogens when used in a simple and rapid PCR assay. One set of oligonucleotide sequences are specific for identifying all but one subspecies of A. avenae; other sets are useful for selectively and specifically identifying either the highly destructive watermelon fruit blotch pathogen, A. avenae subsp. citrulli or the strains of A. avenae subsp. avenaepathogenic for rice. These two subspecies avenaeand citrulli are phenotypically very similar and are very difficult to differentiate. In addition, two other sets of primers and probes specific for identifying A. avenae subsp. citrulli have been designed for the TaqMan (Perkin Elmer) 7700 detection system. One of these primers is the same as described for detecting A. avenae subsp. citrulli by classical PCR, whereas the other primers and probe are different.
In accordance with this discovery, it is an object of the invention to provide the novel oligonucleotides for use as primers for PCR assays for the specific detection and identification of plant pathogenic subspecies of A. avenae. 
It is also an object of the invention to provide primers for the specific detection and identification of the watermelon fruit blotch pathogen, A. avenae subsp. citrulli, thereby differentiating A. avenae subsp. citrulli from other A. avenae subspecies.
It is an additional object of the invention to provide primers for the specific detection and identification of the strains of A. avenae subsp. avenaepathogenic for rice, thereby differentiating the rice strains of A. avenae subsp. avenaefrom A. avenae subsp. avenaeoriginating from other plants and from other subspecies of A. avenae. 
It is another object of the invention to provide PCR assay methods utilizing the novel primers.
It is a further object of the invention to provide a screening method for distinguishing subspecies of A. avenae by utilizing two sets of primers.
It is yet an additional object of the invention to provide a method for assaying seeds for presence of A. avenae subsp. citrulli and for monitoring seed treatment protocols utilizing the novel primers.
It is still another object of the invention to provide a method for assaying seeds for presence of strains of A. avenae subsp. avenaethat are pathogenic for rice and for monitoring seed treatment protocols utilizing the novel primers.
It is an added object of the invention to provide a kit for use in the detection of A. avenae subspec. citrulli. 
It is another added object of the invention to provide a kit for use in the detection of the rice strains of A. avenae subspec. avenae.
Other objects and advantages of the invention will become readily apparent from the following description.